Gram-negative osteomyelitis: clinical and microbiological profile

Introduction: Despite the growing interest in the study of Gram-negative bacilli (GNB) infections, very little information on osteomyelitis caused by GNB is available in the medical literature. Objectives and methods: To assess clinical and microbiological features of 101 cases of osteomyelitis caused by GNB alone, between January 2007 and January 2009, in a reference center for the treatment of high complexity traumas in the city of Sao Paulo. Results: Most patients were men (63%), with median age of 42 years, affected by chronic osteomyelitis (43%) or acute osteomyelitis associated to open fractures (32%), the majority on the lower limbs (71%). The patients were treated with antibiotics as inpatients for 40 days (median) and for 99 days (median) in outpatient settings. After 6 months follow-up, the clinical remission rate was around 60%, relapse 19%, amputation 7%, and death 5%. Nine percent of cases were lost to follow-up. A total of 121 GNB was isolated from 101 clinical samples. The most frequently isolated pathogens were Enterobacter sp. (25%), Acinetobacter baumannii (21%) e Pseudomonas aeruginosa (20%). Susceptibility to carbapenems was about 100% for Enterobacter sp., 75% for Pseudomonas aeruginosa and 60% for Acinetobacter baumannii. Conclusion: Osteomyelitis caused by GNB remains a serious therapeutic challenge, especially when associated to nonfermenting bacteria. We emphasize the need to consider these agents in diagnosed cases of osteomyelitis, so that an ideal antimicrobial treatment can be administered since the very beginning of the therapy. (C) 2012 Elsevier Editora Ltda. All rights reserved.

Healthcare-associated infection in hematopoietic stem cell transplantation patients: risk factors and impact on outcome

Objective: The objective of this study was to analyze the incidence of and risk factors for healthcare-associated infections (HAI) among hematopoietic stem cell transplantation (HSCT) patients, and the impact of such infections on mortality during hospitalization. Methods: We conducted a 9-year (2001-2009) retrospective cohort study including patients submitted to HSCT at a reference center in Sao Paulo, Brazil. The incidence of HAI was calculated using days of neutropenia as the denominator. Data were analyzed using EpiInfo 3.5.1. Results: Over the 9-year period there were 429 neutropenic HSCT patients, with a total of 6816 days of neutropenia. Bloodstream infections (BSI) were the most frequent infection, presenting in 80 (18.6%) patients, with an incidence of 11.7 per 1000 days of neutropenia. Most bacteremia was due to Gram-negative bacteria: 43 (53.8%) cases were caused by Gram-negative species, while 33 (41.2%) were caused by Gram-positive species, and four (5%) by fungal species. Independent risk factors associated with HAI were prolonged neutropenia (odds ratio (OR) 1.07, 95% confidence interval (CI) 1.04-1.10) and duration of fever (OR 1.20, 95% CI 1.12-1.30). Risk factors associated with death in multivariate analyses were age (OR 1.02, 95% CI 1.01-1.43), being submitted to an allogeneic transplant (OR 3.08, 95% CI 1.68-5.56), a microbiologically documented infection (OR 2.96, 95% CI 1.87-4.6), invasive aspergillosis disease (OR 2.21, 95% CI 1.1-4.3), and acute leukemias (OR 2.24, 95% CI 1.3-3.6). Conclusions: BSI was the most frequent HAI, and there was a predominance of Gram-negative microorganisms. Independent risk factors associated with HAI were duration of neutropenia and fever, and the risk factors for a poor outcome were older age, type of transplant (allogeneic), the presence of a microbiologically documented infection, invasive aspergillosis, and acute leukemia. Further prospective studies with larger numbers of patients may confirm the role of these risk factors for a poor clinical outcome and death in this transplant population. (C) 2012 Published by Elsevier Ltd on behalf of International Society for Infectious Diseases.

Chemical resistance of the gram-negative bacteria to different sanitizers in a water purification system

Abstract Background Purified water for pharmaceutical purposes must be free of microbial contamination and pyrogens. Even with the additional sanitary and disinfecting treatments applied to the system (sequential operational stages), Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas alcaligenes, Pseudomonas picketti, Flavobacterium aureum, Acinetobacter lowffi and Pseudomonas diminuta were isolated and identified from a thirteen-stage purification system. To evaluate the efficacy of the chemical agents used in the disinfecting process along with those used to adjust chemical characteristics of the system, over the identified bacteria, the kinetic parameter of killing time (D-value) necessary to inactivate 90% of the initial bioburden (decimal reduction time) was experimentally determined. Methods Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas alcaligenes, Pseudomonas picketti, Flavobacterium aureum, Acinetobacter lowffi and Pseudomonas diminuta were called in house (wild) bacteria. Pseudomonas diminuta ATCC 11568, Pseudomonas alcaligenes INCQS , Pseudomonas aeruginosa ATCC 15442, Pseudomonas fluorescens ATCC 3178, Pseudomonas picketti ATCC 5031, Bacillus subtilis ATCC 937 and Escherichia coli ATCC 25922 were used as 'standard' bacteria to evaluate resistance at 25°C against either 0.5% citric acid, 0.5% hydrochloric acid, 70% ethanol, 0.5% sodium bisulfite, 0.4% sodium hydroxide, 0.5% sodium hypochlorite, or a mixture of 2.2% hydrogen peroxide (H2O2) and 0.45% peracetic acid. Results The efficacy of the sanitizers varied with concentration and contact time to reduce decimal logarithmic (log10) population (n cycles). To kill 90% of the initial population (or one log10 cycle), the necessary time (D-value) was for P. aeruginosa into: (i) 0.5% citric acid, D = 3.8 min; (ii) 0.5% hydrochloric acid, D = 6.9 min; (iii) 70% ethanol, D = 9.7 min; (iv) 0.5% sodium bisulfite, D = 5.3 min; (v) 0.4% sodium hydroxide, D = 14.2 min; (vi) 0.5% sodium hypochlorite, D = 7.9 min; (vii) mixture of hydrogen peroxide (2.2%) plus peracetic acid (0.45%), D = 5.5 min. Conclusion The contact time of 180 min of the system with the mixture of H2O2+ peracetic acid, a total theoretical reduction of 6 log10 cycles was attained in the water purified storage tank and distribution loop. The contact time between the water purification system (WPS) and the sanitary agents should be reviewed to reach sufficient bioburden reduction (over 6 log10).

Antimicrobial effectiveness of silver nanoparticles co-stabilized by the bioactive copolymer pluronic F68

Abstract Background Silver nanoparticles (AgNps) have attracted much interest in biomedical engineering, since they have excellent antimicrobial properties. Therefore, AgNps have often been considered for incorporation into medical products for skin pathologies to reduce the risk of contamination. This study aims at evaluating the antimicrobial effectiveness of AgNps stabilized by pluronic™ F68 associated with other polymers such as polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP). Methods AgNps antimicrobial activity was evaluated using the minimum inhibitory concentration (MIC) method. The action spectrum was evaluated for different polymers associated with pluronic™ F68 against the gram negative bacteria P. aeuroginosa and E. coli and the gram positive bacteria S. Aureus. Results AgNps stabilized with PVP or PVA and co-stabilized with pluronic™ F68 are effective against E. coli and P. aeruginosa microorganisms, with MIC values as low as 0.78% of the concentration of the original AgNps dispersion. The antimicrobial action against S. aureus is poor, with MIC values not lower than 25%. Conclusions AgNps stabilized by different polymeric systems have shown improved antimicrobial activity against gram-negative microorganisms in comparison to unstabilized AgNps. Co-stabilization with the bioactive copolymer pluronic™ F68 has further enhanced the antimicrobial effectiveness against both microorganisms. A poor effectiveness has been found against the gram-positive S. aureus microorganism. Future assays are being delineated targeting possible therapeutic applications.

HPV clearance in postpartum period of HIV-positive and negative women: a prospective follow-up study

Abstract Background HPV persistence is a key determinant of cervical carcinogenesis. The influence of postpartum on HPV clearance has been debated. This study aimed to assess HPV clearance in later pregnancy and postpartum among HIV-positive and negative women. Methods We conducted a follow-up study with 151 HPV-positive women coinfected with HIV, in 2007–2010. After baseline assessment, all women were retested for HPV infection using PCR in later pregnancy and after delivery. Multivariable logistic regressions assessed the putative association of covariates with HPV status in between each one of the successive visits. Results Seventy-one women (47%) have eliminated HPV between the baseline visit and their second or third visits. HIV-positive women took a significantly longer time (7.0 ± 3.8 months) to clear HPV, compared to those not infected by HIV (5.9 ± 3.0 months). HPV clearance was significantly more likely to take place after delivery than during pregnancy (84.5% x 15.5%). Conclusions Both HIV-positive and negative women presented a significant reduction in HPV infection during the postpartum period. HIV-positive status was found to be associated with a longer period of time to clear HPV infection in pregnant women.

Proinflammatory profile of in vitro monocytes in the ageing is affected by lymphocytes presence

Background: Aging is associated with complex and constant remodeling of the immune function, resulting in an increasing susceptibility to infection and others diseases. The infections caused by Gram-negative microorganisms, present in nursing homes and hospitals, constitute one of the most common infections in the elderly, and are mainly combated by innate immune cells. Although the functions of innate immunity seem more preserved during aging than of adaptive immune mechanisms, two systems operate in an integrated way in the body, so that injury in one part of the immune system inevitably affects the other as they are part of a defensive network. The aim of this study was to investigate the in vitro production of proinflammatory (TNF-α, IL-6, IL-1α, CXCL-8 and MCP-1) and antiinflammatory (TGF-α and IL-10) cytokines by monocytes, stimulated or not (basal) with lipopolysaccharide, from healthy young and elderly subjects. By means of PBMCs, we also studied if cytokine profile is altered in these different patient groups, in the presence of lymphocytes, under the same experimental conditions. Results: The monocytes from elderly presented higher basal production of TNF-α, MCP-1 and lower of TGF-α than young monocytes. PBMC showed similar cytokines production, irrespective age or stimulation presence. In the presence of lymphocytes, the spontaneous production of IL-10 was higher and of TGF-α was lower than monocytes, regardless of age. After LPS-stimulation, the presence of lymphocytes resulted in increased IL-6, IL-1α, MCP-1 and IL-10 and decreased CXCL-8 and TGF-α in comparison to pure culture of monocytes from young patients. With age, the same differences were observed, except for CXCL-8 and TGF-α which production was the same between monocytes and PBMC stimulated with LPS.

Questionnaire for monitoring auditory and language development in the first year of life

Purpose: To validate a monitoring questionnaire about hearing and language development applied by community health agents in the first year of life. Methods: Seventy six community health agents, previously trained on infant hearing health, administered a questionnaire to the families of 304 children with ages from 0 to 1 year. The questionnaire contains questions regarding hearing and language development and, for all age groups, the question “Does your child hear well?” was presented. The validity of the questionnaire was assessed by analyzing false positive and false negative rates of the identified children. A double-blind study was conducted so that all children assessed by the questionnaire were submitted to hearing evaluation performed by audiologists. Results: Four children (1.32%) were diagnosed with sensorineural hearing loss (two unilateral), and 69 (22.7%) with conductive hearing loss. The monitoring questionnaire showed specificity of 96% and sensitivity of 67%, with a false-negative rate of 33% for not identifying the unilateral hearing loss, and a false-positive rate of 4%. Conclusion: The questionnaire used has shown to be feasible and relevant to actions of the community health agents of the Family Health Strategy program, with high specificity and moderate sensitivity. The use of the validated instrument should be considered to complement Newborn Hearing Screening Programs, in order to identify late onset or acquired hearing loss.

MRKAd5 HIV-1 Gag/Pol/Nef Vaccine-Induced T-Cell Responses Inadequately Predict Distance of Breakthrough HIV-1 Sequences to the Vaccine or Viral Load

Background: The sieve analysis for the Step trial found evidence that breakthrough HIV-1 sequences for MRKAd5/HIV-1 Gag/Pol/Nef vaccine recipients were more divergent from the vaccine insert than placebo sequences in regions with predicted epitopes. We linked the viral sequence data with immune response and acute viral load data to explore mechanisms for and consequences of the observed sieve effect. Methods: Ninety-one male participants (37 placebo and 54 vaccine recipients) were included; viral sequences were obtained at the time of HIV-1 diagnosis. T-cell responses were measured 4 weeks post-second vaccination and at the first or second week post-diagnosis. Acute viral load was obtained at RNA-positive and antibody-negative visits. Findings: Vaccine recipients had a greater magnitude of post-infection CD8+ T cell response than placebo recipients (median 1.68% vs 1.18%; p = 0.04) and greater breadth of post-infection response (median 4.5 vs 2; p = 0.06). Viral sequences for vaccine recipients were marginally more divergent from the insert than placebo sequences in regions of Nef targeted by pre-infection immune responses (p = 0.04; Pol p = 0.13; Gag p = 0.89). Magnitude and breadth of pre-infection responses did not correlate with distance of the viral sequence to the insert (p. 0.50). Acute log viral load trended lower in vaccine versus placebo recipients (estimated mean 4.7 vs 5.1) but the difference was not significant (p = 0.27). Neither was acute viral load associated with distance of the viral sequence to the insert (p>0.30). Interpretation: Despite evidence of anamnestic responses, the sieve effect was not well explained by available measures of T-cell immunogenicity. Sequence divergence from the vaccine was not significantly associated with acute viral load. While point estimates suggested weak vaccine suppression of viral load, the result was not significant and more viral load data would be needed to detect suppression.

Brazilian spotted fever: Real-time PCR for diagnosis of fatal cases

Suspicion of Brazilian spotted fever (BSF) should occur in endemic regions upon surveillance of the acute febrile icteric hemorrhagic syndrome (AFIHS). However, limitations associated with currently available laboratory tests pose a challenge to early diagnosis, especially in fatal cases. Two real-time PCR (qPCR) protocols were evaluated to diagnose BSF in 110 fatal AFIHS cases, collected in BSF-endemic regions in 2009-2010. Of these, 24 were positive and 86 negative by indirect immunofluorescence (IFA) assay (cutoff IgG and/or IgM >= 128). DNA from these samples was used in the qPCR protocols: one to detect Rickettsia spp. (Citrate synthase gene) and another to determine spotted fever group (SFG) Rickettsia species (OmpA gene). Of the 24 IFA-positive samples, 5 (21%) were positive for OmpA and 9 (38%) for citrate synthase. In the IFA-negative group (n = 86), OmpA and citrate synthase were positive in 23 (27%) and 27 (31%), respectively. These results showed that the 2 qPCR protocols were about twice as sensitive as the IFA test alone (93% concordance). In conclusion, qPCR is a sensitive method for the diagnosis of fatal BSF cases and should be considered for routine surveillance of AFIHS in places like Brazil, where spotted fever-related lethality is high and other endemic diseases like dengue and leptospirosis can mislead diagnosis. (C) 2012 Elsevier GmbH. All rights reserved.

Evaluation of reference-based two-color methods for measurement of gene expression ratios using spotted cDNA microarrays

Abstract Background Spotted cDNA microarrays generally employ co-hybridization of fluorescently-labeled RNA targets to produce gene expression ratios for subsequent analysis. Direct comparison of two RNA samples in the same microarray provides the highest level of accuracy; however, due to the number of combinatorial pair-wise comparisons, the direct method is impractical for studies including large number of individual samples (e.g., tumor classification studies). For such studies, indirect comparisons using a common reference standard have been the preferred method. Here we evaluated the precision and accuracy of reconstructed ratios from three indirect methods relative to ratios obtained from direct hybridizations, herein considered as the gold-standard. Results We performed hybridizations using a fixed amount of Cy3-labeled reference oligonucleotide (RefOligo) against distinct Cy5-labeled targets from prostate, breast and kidney tumor samples. Reconstructed ratios between all tissue pairs were derived from ratios between each tissue sample and RefOligo. Reconstructed ratios were compared to (i) ratios obtained in parallel from direct pair-wise hybridizations of tissue samples, and to (ii) reconstructed ratios derived from hybridization of each tissue against a reference RNA pool (RefPool). To evaluate the effect of the external references, reconstructed ratios were also calculated directly from intensity values of single-channel (One-Color) measurements derived from tissue sample data collected in the RefOligo experiments. We show that the average coefficient of variation of ratios between intra- and inter-slide replicates derived from RefOligo, RefPool and One-Color were similar and 2 to 4-fold higher than ratios obtained in direct hybridizations. Correlation coefficients calculated for all three tissue comparisons were also similar. In addition, the performance of all indirect methods in terms of their robustness to identify genes deemed as differentially expressed based on direct hybridizations, as well as false-positive and false-negative rates, were found to be comparable. Conclusion RefOligo produces ratios as precise and accurate as ratios reconstructed from a RNA pool, thus representing a reliable alternative in reference-based hybridization experiments. In addition, One-Color measurements alone can reconstruct expression ratios without loss in precision or accuracy. We conclude that both methods are adequate options in large-scale projects where the amount of a common reference RNA pool is usually restrictive.

Synthesis and Photodynamic Effect of New Highly Photostable Decacationically Armed [60]- and [70]Fullerene Decaiodide Monoadducts To Target Pathogenic Bacteria and Cancer Cells

Novel water-soluble decacationically armed C-60 and C-70 decaiodide monoadducts, C-60- and C-70[>M(C3N6+C3)(2)], were synthesized, characterized, and applied as photosensitizers and potential nano-PDT agents against pathogenic bacteria and cancer cells. A high number of cationic charges per fullerene cage and H-bonding moieties were designed for rapid binding to the anionic residues displayed on the outer parts of bacterial cell walls. In the presence of a high number of electron-donating iodide anions as parts of quaternary ammonium salts in the arm region, we found that C-70[>M(C3N6+C3)(2)] produced more HO center dot than C-60[>M(C3N6+C3)(2)], in addition to O-1(2). This finding offers an explanation of the preferential killing of Gram-positive and Gram-negative bacteria by C-60[>M(C3N6+C3)(2)] and C-70[>M(C3N6+C3)(2)], respectively. The hypothesis is that O-1(2) can diffuse more easily into porous cell walls of Gram-positive bacteria to reach sensitive sites, while the less permeable Gram-negative bacterial cell wall needs the more reactive HO center dot to cause real damage.

Lactobacillus sakei 2a and its Concentrated Acid Extract on Inhibition of Food-Borne Salmonella strains

Lactic acid bacteria are used in food production to provide desirable organoleptic characteristics, and can also act as biopreservatives, controlling the growth of undesirable microorganisms. In this study, we examined the antimicrobial action of Lactobacillus sakei 2a and its concentrated acid extract against food-borne Salmonella spp. The extract was obtained by acid extraction from culture broth of L. sakei 2a and was designated extract 2a. We determined that extract 2a had significant activity (approximately 500 AU ml(-1)). We used different antimicrobial substances alone or in combination with extract 2a to evaluate the inhibitory activity of the various treatments on a pool of five Salmonella strains. The pathogen Listeria monocytogenes Scott A Cm-r Em(r) was used as an indicator strain of inhibitory activity. In summary, all antimicrobials substances that were tested showed an inhibitory effect against the growth of Salmonella, andthis action was enhanced in the presence of extract 2a. Moreover, among the treatments applied, the combination of extract 2a and 0.1% lactic acid exhibited the most potent inhibitory effect towards the pool of Salmonella strains. Our findings indicate that L. sakei 2a and extract 2a, especially in combination with other antimicrobials, present potential technological application in the control of salmonellae in foods.

Functional and structural studies of the disulfide isomerase DsbC from the plant pathogen Xylella fastidiosa reveals a redox-dependent oligomeric modulation in vitro

Xylella fastidiosa is a Gram-negative bacterium that grows as a biofilm inside the xylem vessels of susceptible plants and causes several economically relevant crop diseases. In the present study, we report the functional and low-resolution structural characterization of the X. fastidiosa disulfide isomerase DsbC (XfDsbC). DsbC is part of the disulfide bond reduction/isomerization pathway in the bacterial periplasm and plays an important role in oxidative protein folding. In the present study, we demonstrate the presence of XfDsbC during different stages of X. fastidiosa biofilm development. XfDsbC was not detected during X. fastidiosa planktonic growth; however, after administering a sublethal copper shock, we observed an overexpression of XfDsbC that also occurred during planktonic growth. These results suggest that X. fastidiosa can use XfDsbC in vivo under oxidative stress conditions similar to those induced by copper. In addition, using dynamic light scattering and small-angle X-ray scattering, we observed that the oligomeric state of XfDsbC in vitro may be dependent on the redox environment. Under reducing conditions, XfDsbC is present as a dimer, whereas a putative tetrameric form was observed under nonreducing conditions. Taken together, our findings demonstrate the overexpression of XfDsbC during biofilm formation and provide the first structural model of a bacterial disulfide isomerase in solution. Structured digital abstract XfDsbC and XfDsbC bind by x ray scattering (View Interaction: 1, 2) XfDsbC and XfDsbC bind by molecular sieving (View interaction) XfDsbC and XfDsbC bind by comigration in non denaturing gel electrophoresis (View interaction) XfDsbC and XfDsbC bind by cross-linking study (View Interaction: 1, 2) XfDsbC and XfDsbC bind by dynamic light scattering (View Interaction: 1, 2)

Sardinian goat's milk as source of bacteriocinogenic potential protective cultures

Goat breeding in Sardinia constitutes an important source of income for farming and shepherding activities. In this study 170 LAB strains were isolated from Sardinian goat's milk and tested for bacteriocins production against several food-borne pathogenic microorganisms. Four isolates (SD1, SD2, SD3 and SD4) were selected for their effective inhibition on Listeria monocytogenes. The strains were classified as members of Enterococcus genus, according to their biochemical and physiological characteristics, and then genetically identified as Enterococcus faecium. In MRS broth at 37 degrees C, bacteriocins SD1 and SD2 were produced at much higher levels (51200 AU/ml) compared to bacteriocin SD3 (3200 AU/ml) and bacteriocin SD4 (800 AU/ml). Their peptides were inactivated by proteolytic enzymes, but not when treated with alpha-amylase, catalase and lipase. The four bacteriocins remained stable at pH from 2.0 to 12.0, after exposure to 100 degrees C for 120 min and were not affected by the presence of surfactants and salts (N-Laourylsarcosine, NaCl, SDS, Triton X-100, Tween 20, Tween 80 and urea). Their molecular size was determined to be approximately 5 kDa by tricine-SDS-PAGE. Since the strains exhibited a strong antimicrobial activity against 21 L monocytogenes strains and 6 Salmonella spp. isolates, they should be considered as potential bio-preservatives cultures for fermented food productions. Moreover, due to their technological features, the four strains could be taken in account for using as adjunct NSLAB (non-starter lactic acid bacteria) rather than as starter culture. (C) 2011 Elsevier Ltd. All rights reserved.

Coordinated expression of oestrogen and androgen receptors in HER2-positive breast carcinomas: impact on proliferative activity

Aims Human epidermal growth factor receptor 2 (HER2)-positive breast cancers are aggressive neoplasms associated with a variable response to systemic therapies. Therefore, the identification of biomarkers to better characterise this heterogeneity would improve treatment efficacy. The aim of this study was to evaluate the influence of androgen receptor (AR) and oestrogen receptor (ER) on clinicopathological features in a series of HER2-positive breast carcinomas. Methods A total of 104 carcinomas were selected and reviewed. Immunohistochemical studies for ER, progesterone receptor and Ki-67 were analysed on tumour whole histological sections. AR expression was analysed on samples represented on tissue microarrays. According to steroid receptor expression, cases were classified into three groups: AR positive/ER positive (48 cases), AR positive/ER negative (41 cases) and AR negative/ER negative (13 cases). Results AR-positive tumours corresponded to 89 (85.6%) of 104 carcinomas. AR-positive carcinomas were associated with a higher frequency of ER and progesterone receptor co-expression and lower proliferative activity determined by the expression of Ki-67. AR-negative carcinomas were more often high grade. The group of AR-positive/ER-negative carcinomas was associated with the highest frequency of apocrine morphological features. The group of AR-negative/ER-negative carcinomas was associated with the highest proliferative activity and the highest frequency of high histological and nuclear grade. The lowest frequency of high-grade tumours and the lowest proliferative activity were seen among tumours with expression of both receptors. Conclusions These results suggest that co-expression of AR and ER can provide a protective effect based on phenotypical presentation of HER2-positive carcinomas. Furthermore, lack of both steroid hormone receptors characterises the most aggressive phenotype.